Maintenance of genome stability is essential for cell survival and is critical for the prevention of carcinogenesis. Indeed, genetic instability is one of the key features of cancer cells. Because errors in DNA replication are a major cause of genomic instability, it is critical to understand all aspects involved in the regulation of DNA replication. However, relatively few studies have addressed how chromatin structure, the physiological organization of DNA in eukaryotic cells, impacts and regulates the DNA replication process. During DNA replication, chromatin must be temporarily disassembled to allow the DNA replication machinery access to the DNA template. The newly-synthesized DNA strands are then immediately reassembled into their original chromatin state. Uncoupling of DNA synthesis and chromatin assembly results in genome instability. Because of the critical nature of genome stability, it is important to understand how DNA synthesis is coupled to chromatin assembly. We use the yeast Saccharomyces cerevisiae as a eukaryotic model organism to address questions regarding this essential, but poorly understood process. Recent studies have implicated the acetylation of lysine 56 on histone H3 (H3-K56) as a key regulatory event during the DNA synthesis and chromatin assembly. Acetylation of H3-K56 occurs transiently during S phase. Cells with altered acetylation of H3-K56 are highly sensitive to DNA damaging agents, suggesting that this modification is important for maintaining genome stability. The main objective of this proposal is to test the hypothesis that acetylation of H3-K56 by a novel and unique HAT Rtt109, which we have recently identified, maintains genome integrity by coupling DNA replication with the assembly of newly-replicated DNA into nucleosomes mediated by histone chaperones. Results from these studies will provide mechanistic insights into the role of histone modifications in coupling DNA synthesis with nucleosome assembly, an important aspect in the maintenance of genome stability and thus normal cell functions.